Calponin interacts with several Ca2+ binding proteins in a Ca(2+)-dependent manner. In order to determine the possible biological relevance of these interactions in smooth muscle function, it is necessary to characterize the strength and stoichiometry of the complexes formed. The interaction between calponin and calmodulin can be monitored through an acrylodan label on a cysteine of calponin. The fluorescently labeled calponin possesses the same biological function and physical behavior in binding to calmodulin as the native calponin. This probe is very environment-sensitive and responds to the calponin-calmodulin interaction by the emission peak blue-shifting 20 nm and by the fluorescent quantum yield increasing 3.5 times at 460 nm. The stoichiometric nature of this complex has been determined using analytical ultracentrifugation and is two calmodulins to one calponin, and the interaction is Ca(2+)-sensitive with a Kd1 of < or = 0.22 microM and a Kd2 of 2.5-3.4 microM. Calmodulin is not the only protein which interacts with calponin in this manner, but rather this interaction seems to be a general feature attributable to all hydrophobic patch exposing proteins, suggesting that it may be nonspecific, occurring because of a generalized mode of interaction. Two other proteins, S-100b from bovine brain and SMCaBP-11 from smooth muscle, had stronger affinities for calponin, and in particular interaction of SMCaBP-11 with calponin may be biologically relevant. In determining the nature of calponin's interaction with these Ca2+ binding proteins, it was apparent there was no effect of Ca2+ upon calponin itself and physical studies could find no evidence that calponin interacts with calcium.

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcγ-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcγ-receptor-mediated nitric oxide (86.0 ± 8.3%) and TNF (73.1 ± 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcγ-receptor-induced IL-1β release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.

Interferon-gamma (IFN-gamma) is an important regulatory cytokine in cell proliferation, differentiation, adhesion, mediator release, and gene induction. This diversity of effector roles is achieved by a variety of incompletely understood mechanisms. In the mast cell (MC), IFN-gamma downregulates mediator synthesis and secretion. The present study demonstrates and characterizes for the first time IFN-gamma inhibition of adhesion of the MC analogue RBL-2H3 to the extracellular matrix protein fibronectin (FN). Inhibition requires preincubation of the cells with IFN-gamma for 20 hr, and is statistically significant at 100 U/ml IFN-gamma. Flow cytometry indicates that cell surface expression of very late antigen-4 (VLA-4), VLA-5, and the vitronectin receptor (VNR) remain constant following IFN-gamma treatment, indicating the inhibitory effect of IFN-gamma on adhesion to FN is not achieved through a reduction in integrin receptors for FN. Fluorescent labelling with Texas red phalloidin demonstrated rearrangement of the actin cytoskeleton in response to IFN-gamma was not significant. The tyrosine phosphatase inhibitor vanadate, and the nitric oxide (NO) synthase inhibitor L-NAME, reduced the IFN-gamma effect on adhesion to FN by 62 and 70%, respectively, demonstrating that the IFN-gamma effect is dependent upon the production of NO, potentially though a tyrosine phosphatase dependent mechanism. The NO donors sodium nitroprusside and S-nitrosoglutathione mimicked the effect of IFN-gamma. Thus, following stimulation with IFN-gamma, NO plays an autocrine role in the MC, and is able to modulate integrin function. This adds to the pathways NO is able to inhibit in the mast cell, shows that endogenous NO is able to inhibit these pathways, and suggests NO is impinging upon an element common to many signalling mechanisms in the MC.

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer: 1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD).(ABSTRACT TRUNCATED AT 250 WORDS)

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that C P 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca'+-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxylterminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.

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Nitric oxide (NO) is a potent mediator synthesized by a variety of cells involved in inflammatory reactions. We investigated the expression of NO synthase (NOS) in rat peritoneal mast cells (PMC). Small amounts of eNOS mRNA were detected basally, whereas neither mRNA for iNOS nor nNOS was detected in unstimulated PMC. Following stimulation by antigen, interferon-γ (IFN-γ), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-γ, or anti-CD8, nitrite production was increased significantly (8.4±0.6, 7.6±0.9, and 6.6±0.9 μM/2×105 cells/48 h NO2−, respectively; P&lt;0.01), whereas unstimulated PMC released 2.1 ± 0.3 μM/2 × 105 cells/48 h NO2−. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.